Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein–protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed “whorls” because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.


Background
Gap junctions are clusters of intercellular channels that directly connect the cytoplasm of adjacent cells, providing a conduit for the exchange of metabolites and ions.In chordates, gap junctions are formed by connexins, a diverse family of membrane proteins that have the ability to oligomerize into dodecameric channels.Mutations in connexin genes are the cause of a variety of inherited diseases.In many cases, these pathologies have been linked to trafficking defects that compromise the ability of the channel to form functional gap junctions [1,2].Among the 21 connexins that have been identified in humans, only a few variants have been studied in terms of trafficking through intracellular compartments [2][3][4].To study basic transport mechanisms of gap junction proteins, researchers take advantage of expression systems, such as HeLa or HEK293T cells, and transfect these cells with recombinant expression vectors.In this article, we describe a detailed protocol that can be used to determine endoplasmic reticulum (ER) retention defects for connexin 36 (Cx36).In a previous study, we described a transport defect that prevented the functional ER export of Cx36, causing the connexin to accumulate in the ER [5].This retention mechanism promoted the formation of gap junction-like aggregates that reshaped the ER into concentric multimembrane vesicles (Figures 1B and D), which we termed connexin whorls.These structures were characterized by several distinct features: 1) Each sheet within the whorl exhibited ultrastructural features that were indistinguishable from an actual gap junction; 2) whorls are hollow inside and their diameter varied, ranging from 0.3 to 3 µm; 3) whorls colocalized with ER-phagy receptors Tex264 (Testis-expressed protein 264) (Figure 1C) and p62; 4) whorl formation requires docking interactions of extracellular loops in Cx36 facing the ER lumen.Substituting the extracellular loop cysteines (C55 or C62) via site-directed mutagenesis prevents whorl formation.Similar whorllike structures were reported for the lens connexin Cx50 in a previous study by Lichtenstein et al., [6], which suggests that ER-derived whorls are formed by many connexins that oligomerize in ER.Therefore, the protocol we describe here is not only applicable for Cx36 but might be used for other connexin isoforms.However, two important aspects have to be considered: 1) Some connexins, for instance Cx43, oligomerize in the Golgi [7], which would make the docking of Cx43 containing connexons in the ER impossible; and 2) whorls have to be tested for the presence of ER proteins, for instance Tex264, to determine the compartment they originated in (Figure 1C).Based on the previous measurements, determine a size threshold to exclude smaller vesicles from quantification.As described in Tetenborg et al., [5], vesicles with a diameter smaller than 0.3 μm were not considered whorls.2. Open the cell counter plugin (Figure 5) and click initialize.Confocal scans can be opened in ImageJ.Before the quantification, several scans of the experiment should be inspected to determine a size threshold for whorls (see Figure 3).Every vesicle with a diameter under the size threshold is excluded from quantification.The cell counter plugin has to be initialized in order to select vesicles that are counted.Each vesicle that is considered a whorl based on shape and diameter can be selected by a mouse click.

Figure 1 .
Figure 1.Membrane topology of Cx36.Protein structure of Cx36 is illustrated.The two extracellular loops of Cx36 (indicated by upper circle and red arrow) contain three cysteines.The C-terminal tail contains the PDZ binding motif consisting of the following amino acid sequence: SAYV.

Figure 2 .
Figure 2. Connexin whorls.A. Transiently transfected HEK293T cells expressing Cx36.Cx36 is concentrated at gap junctions, indicated by the long arrow.Cx36 is also visible in perinuclear structures resembling the Golgi apparatus.Indicated by the short arrow.Scale: 5 μm.B. Transfected HEK293T cell expressing the trafficking deficient Cx36/S318ter mutant.Cx36 is retained in the ER and accumulates in ER whorls, which are labeled with short arrows.Gap junctions are still formed by the Cx36/S318ter mutant.Labeled with the long arrow.Scale: 5 μm.C. Example of Cx36 whorls colocalizing with the ER phagy receptor Tex264.Scale: 5 μm.Magnified inset: 2.5 μm.D. Cartoon illustrating the composition of ER whorls.E. Whorls are seen for different connexins.Confocal scan of Halo-tagged zebrafish Cx34.7, an orthologue of Cx36.Scale: 10 μm.

3 .
Select a type of counter and count the whorls in your stack via left clicks.

Figure 4 . 8 Published: xxx xx, 2024 Figure 5 .
Figure 4. Measurement of whorl diameter using ImageJ.A. 1) A region of interest (ROI) surrounding the whorl can be selected using the rectangular selection tool.The ROI can be duplicated (shift + D). 2) To measure the diameter of a selected whorl, draw a line using the line selection tool and 3) use the measure function (Ctrl + M).B. The length will be displayed in the last column.
This protocol or parts of it has been used and validated in the following research article(s): Tetenborg et al., (2023) Journal of Biological Chemistry (Figure 1, panel C-E, Quantification of 11-30 vesicles, from 9-11 cell clusters)